Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Parsing ß-catenin's cell adhesion and Wnt signaling functions in malignant mammary tumor progression.

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression.

Heterogeneity within a tumour increases its ability to adapt to constantly changing constraints, but adversely affects a patient's prognosis, therapy response and clinical outcome. Intratumoural heterogeneity results from a combination of extrinsic factors from the tumour microenvironment and intrinsic parameters from the cancer cells themselves, including their genetic, epigenetic and transcriptomic traits, their ability to proliferate, migrate and invade, and their stemness and plasticity attributes. Cell plasticity constitutes the ability of cancer cells to rapidly reprogramme their gene expression repertoire, to change their behaviour and identities, and to adapt to microenvironmental cues. These features also directly contribute to tumour heterogeneity and are critical for malignant tumour progression. In this article, we use breast cancer as an example of the origins of tumour heterogeneity (in particular, the mutational spectrum and clonal evolution of progressing tumours) and of tumour cell plasticity (in particular, that shown by tumour cells undergoing epithelial-to-mesenchymal transition), as well as considering interclonal cooperativity and cell plasticity as sources of cancer cell heterogeneity. We review current knowledge on the functional contribution of cell plasticity and tumour heterogeneity to malignant tumour progression, metastasis formation and therapy resistance.

Inferring signalling dynamics by integrating interventional with observational data.

MOTIVATION: In order to infer a cell signalling network, we generally need interventional data from perturbation experiments. If the perturbation experiments are time-resolved, then signal progression through the network can be inferred. However, such designs are infeasible for large signalling networks, where it is more common to have steady-state perturbation data on the one hand, and a non-interventional time series on the other. Such was the design in a recent experiment investigating the coordination of epithelial-mesenchymal transition (EMT) in murine mammary gland cells. We aimed to infer the underlying signalling network of transcription factors and microRNAs coordinating EMT, as well as the signal progression during EMT. RESULTS: In the context of nested effects models, we developed a method for integrating perturbation data with a non-interventional time series. We applied the model to RNA sequencing data obtained from an EMT experiment. Part of the network inferred from RNA interference was validated experimentally using luciferase reporter assays. Our model extension is formulated as an integer linear programme, which can be solved efficiently using heuristic algorithms. This extension allowed us to infer the signal progression through the network during an EMT time course, and thereby assess when each regulator is necessary for EMT to advance. AVAILABILITY AND IMPLEMENTATION: R package at https://github.com/cbg-ethz/timeseriesNEM. The RNA sequencing data and microscopy images can be explored through a Shiny app at https://emt.bsse.ethz.ch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Transgenic MMTV-Flippase Mouse Line for Molecular Engineering in Mammary Gland and Breast Cancer Mouse Models.

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.

NGS-pipe: a flexible, easily extendable and highly configurable framework for NGS analysis.

Motivation: Next-generation sequencing is now an established method in genomics, and massive amounts of sequencing data are being generated on a regular basis. Analysis of the sequencing data is typically performed by lab-specific in-house solutions, but the agreement of results from different facilities is often small. General standards for quality control, reproducibility and documentation are missing. Results: We developed NGS-pipe, a flexible, transparent and easy-to-use framework for the design of pipelines to analyze whole-exome, whole-genome and transcriptome sequencing data. NGS-pipe facilitates the harmonization of genomic data analysis by supporting quality control, documentation, reproducibility, parallelization and easy adaptation to other NGS experiments. Availability and implementation: https://github.com/cbg-ethz/NGS-pipe. Contact: niko.beerenwinkel@bsse.ethz.ch.

Inflammatory Cytokines Induce Podoplanin Expression at the Tumor Invasive Front.

Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-ß, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC.

Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells.

BACKGROUND: The tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood. METHODS: We have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro. RESULTS: Upon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression. CONCLUSION: The identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression.

Foxf2 plays a dual role during transforming growth factor beta-induced epithelial to mesenchymal transition by promoting apoptosis yet enabling cell junction dissolution and migration.

BACKGROUND: The most life-threatening step during malignant tumor progression is reached when cancer cells leave the primary tumor mass and seed metastasis in distant organs. To infiltrate the surrounding tissue and disseminate throughout the body, single motile tumor cells leave the tumor mass by breaking down cell-cell contacts in a process called epithelial to mesenchymal transition (EMT). An EMT is a complex molecular and cellular program enabling epithelial cells to abandon their differentiated phenotype, including cell-cell adhesion and cell polarity, and to acquire mesenchymal features and invasive properties. METHODS: We employed gene expression profiling and functional experiments to study transcriptional control of transforming growth factor (TGF)ß-induced EMT in normal murine mammary gland epithelial (NMuMG) cells. RESULTS: We identified that expression of the transcription factor forkhead box protein F2 (Foxf2) is upregulated during the EMT process. Although it is not required to gain mesenchymal markers, Foxf2 is essential for the disruption of cell junctions and the downregulation of epithelial markers in NMuMG cells treated with TGFß. Foxf2 is critical for the downregulation of E-cadherin by promoting the expression of the transcriptional repressors of E-cadherin, Zeb1 and Zeb2, while repressing expression of the epithelial maintenance factor Id2 and miRNA 200 family members. Moreover, Foxf2 is required for TGFß-mediated apoptosis during EMT by the transcriptional activation of the proapoptotic BH3-only protein Noxa and by the negative regulation of epidermal growth factor receptor (EGFR)-mediated survival signaling through direct repression of its ligands betacellulin and amphiregulin. The dual function of Foxf2 during EMT is underscored by the finding that high Foxf2 expression correlates with good prognosis in patients with early noninvasive stages of breast cancer, but with poor prognosis in advanced breast cancer. CONCLUSIONS: Our data identify the transcription factor Foxf2 as one of the important regulators of EMT, displaying a dual function in promoting tumor cell apoptosis as well as tumor cell migration.

Heterogeneity for IGF-II production maintained by public goods dynamics in neuroendocrine pancreatic cancer.

The extensive intratumor heterogeneity revealed by sequencing cancer genomes is an essential determinant of tumor progression, diagnosis, and treatment. What maintains heterogeneity remains an open question because competition within a tumor leads to a strong selection for the fittest subclone. Cancer cells also cooperate by sharing molecules with paracrine effects, such as growth factors, and heterogeneity can be maintained if subclones depend on each other for survival. Without strict interdependence between subclones, however, nonproducer cells can free-ride on the growth factors produced by neighboring producer cells, a collective action problem known in game theory as the "tragedy of the commons," which has been observed in microbial cell populations. Here, we report that similar dynamics occur in cancer cell populations. Neuroendocrine pancreatic cancer (insulinoma) cells that do not produce insulin-like growth factor II (IGF-II) grow slowly in pure cultures but have a proliferation advantage in mixed cultures, where they can use the IGF-II provided by producer cells. We show that, as predicted by evolutionary game theory, producer cells do not go extinct because IGF-II acts as a nonlinear public good, creating negative frequency-dependent selection that leads to a stable coexistence of the two cell types. Intratumor cell heterogeneity can therefore be maintained even without strict interdependence between cell subclones. Reducing the amount of growth factors available within a tumor may lead to a reduction in growth followed by a new equilibrium, which may explain relapse in therapies that target growth factors.

VEGF receptor-2-specific signaling mediated by VEGF-E induces hemangioma-like lesions in normal and in malignant tissue.

UNLABELLED: Viral VEGF-E (ovVEGF-E), a homolog of VEGF-A, was discovered in the genome of Orf virus. Together with VEGF-A, B, C, D, placental growth factor (PlGF) and snake venom VEGF (svVEGF), ovVEGF-E is a member of the VEGF family of potent angiogenesis factors with a bioactivity similar to VEGF-A: it induces proliferation, migration and sprouting of cultured vascular endothelial cells and proliferative lesions in the skin of sheep, goat and man that are characterized by massive capillary proliferation and dilation. These biological functions are mediated exclusively via its interaction with VEGF receptor-2 (VEGFR-2). Here, we have generated transgenic mice specifically expressing ovVEGF-E in ß-cells of the endocrine pancreas (Rip1VEGF-E; RVE). RVE mice show an increase in number and size of the islets of Langerhans and a distorted organization of insulin and glucagon-expressing cells. Islet endothelial cells of RVE mice hyper-proliferate and form increased numbers of functional blood vessels. In addition, the formation of disorganized lymphatic vessels and increased immune cell infiltration is observed. Upon crossing RVE single-transgenic mice with Rip1Tag2 (RT2) transgenic mice, a well-studied model of pancreatic ß-cell carcinogenesis, double-transgenic mice (RT2;RVE) display hyper-proliferation of endothelial cells resulting in the formation of hemangioma-like lesions. In addition, RT2;RVE mice exhibit activated lymphangiogenesis at the tumor periphery and increased neutrophil and macrophage tumor infiltration and micro-metastasis to lymph nodes and lungs. These phenotypes markedly differ from the phenotypes observed with the transgenic expression of the other VEGF family members in ß-cells of normal mice and of RT2 mice.

Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition.

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.

Junctional adhesion molecule B interferes with angiogenic VEGF/VEGFR2 signaling.

De novo formation of blood vessels is a pivotal mechanism during cancer development. During the past few years, antiangiogenic drugs have been developed to target tumor vasculature. However, because of limitations and adverse effects observed with current therapies, there is a strong need for alternative antiangiogenic strategies. Using specific anti-junctional adhesion molecule (JAM)-B antibodies and Jam-b-deficient mice, we studied the role in antiangiogenesis of JAM-B. We found that antibodies against murine JAM-B, an endothelium-specific adhesion molecule, inhibited microvessel outgrowth from ex vivo aortic rings and in vitro endothelial network formation. In addition, anti-JAM-B antibodies blocked VEGF signaling, an essential pathway for angiogenesis. Moreover, increased aortic ring branching was observed in aortas isolated from Jam-b-deficient animals, suggesting that JAM-B negatively regulates proangiogenic pathways. In mice, JAM-B expression was detected in de novo-formed blood vessels of tumors, but anti-JAM-B antibodies unexpectedly did not reduce tumor growth. Accordingly, JAM-B deficiency in vivo had no impact on blood vessel formation, suggesting that targeting JAM-B in vivo may be offset by other proangiogenic mechanisms. In conclusion, despite the promising effects observed in vitro, targeting JAM-B during tumor progression seems to be inefficient as a stand-alone antiangiogenesis therapy.

An immature B cell population from peripheral blood serves as surrogate marker for monitoring tumor angiogenesis and anti-angiogenic therapy in mouse models.

Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45(dim)VEGFR1(-)CD31(low), that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45(dim)VEGFR1(-)CD31(low) cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45(dim)VEGFR1(-)CD31(low) cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer.

Transcription factor Dlx2 protects from TGFß-induced cell-cycle arrest and apoptosis.

Acquiring resistance against transforming growth factor ß (TGFß)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFß's tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFß-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFß receptor II (TGFßRII) gene expression and reduces canonical, Smad-dependent TGFß signalling and expression of the cell-cycle inhibitor p21(CIP1) and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFß from its tumour suppressive to its tumour-promoting functions.

PI3K signalling is required for a TGFß-induced epithelial-mesenchymal-like transition (EMT-like) in human melanoma cells.

Epithelial to mesenchymal transition (EMT) is a programme defined in epithelial cells and recognized as playing a critical role in cancer progression. Although melanoma is not a cancer of epithelial cells, hallmarks of EMT have been described to play a critical role in melanoma progression. Here, we demonstrate that long-term TGFß exposure can induce a dedifferentiated EMT-like state resembling a previously described invasive phenotype (EMT-like). TGFß-induced EMT-like is marked by the downregulation of melanocyte differentiation markers, such as MITF, and the upregulation of mesenchymal markers, such as N-cadherin, and an increase in melanoma cell migration and cell invasion. Pharmacological interference shows the dependency of TGFß-induced EMT-like on the activation of the PDGF signalling pathway and the subsequent activation of PI3K in human melanoma cells. Together, the data provide novel insights into the transcriptional plasticity of melanoma cells that might contribute to tumor progression in patients and propose avenues to therapeutic interventions.